The long-term objective of this proposal is to study the mechanism of photoreceptor differentiation and outer segment formation and the role of a newly discovered retina-specific protein in these processes. Specific aims are to: l) Test the hypothesis that a novel double-zinc- finger protein, the actin-binding LIM (ab/LIM) protein, plays an essential role in retinal development and/or photoreceptor maintenance. 2) Identify additional members of the intracellular signaling pathway in the retina in which ab/LIM protein may be involved. 3) Characterize the modulation of ab/LIM function by phosphorylation. 4) Determine the gene locus of ab/LIM as a first step in assessing the potential of ab/LIM as a candidate gene in hereditary retinal diseases. In order to achieve these objectives we will use molecular biological methods to perturb ab/LIM expression in vivo within transgenic mouse retinas. Based on the primary structure of the protein, we will generate mouse strains that either express dominantly- acting mutant forms of ab/LIM or we will selectively eliminate the retina- specific expression of ab/LIM protein. To identify members of the putative ab/LIM signaling pathway we will use both molecular biological methods to identify ab/LIM binding partners expressed in eukaryotic cells in vivo and biochemical methods to detect ab/LIM interactions with other proteins in retinal extracts in vitro. Since we know that massive phosphorylation of ab/LIM is apparently the normal state in adult light-adapted retinas, we will check the possibility that the state of ab/LIM phosphorylation is under physiological regulation during development, at different times of the light/dark cycle, or after exposure to bright light. Finally, the specific expression of one isoform of ab/LIM protein in the retina, with particular enrichment in photoreceptor cells, raises the possibility that this protein might be involved in hereditary retinal diseases. Mapping the subchromosomal locus for the ab/LIM gene will allow us to begin consideration of ab/LIM as a candidate gene in specific forms of retinal disease. Taken together, these lines of research are aimed at defining the role of a newly discovered retinal protein in both normal retinal development and, when altered, in hereditary retinal diseases.